Investigation of the impact of αCD45 cellular backpacks on the mechanobiology of T cell activation and tumor cell cross-talk

Abstract

Immune checkpoint blockade-resistant solid tumors present a major therapeutic challenge. Although adoptive T cell therapies, such as CAR T cells, have shown remarkable success in treating hematological malignancies like leukemia, they have not yet achieved comparable efficacy in solid tumors. In the majority of patients with solid tumors, transferred T cells rapidly lose their functional phenotype following adoptive transfer, leaving them vulnerable to the immunosuppressive tumor microenvironment (TME) [#zhao2019TcelltherapyReview]. Considering the significant promise of adoptive T cell therapies, there is intense interest in developing strategies to enhance their persistence and function within solid tumors. Recent work under review for publishing in the Mitragotri lab has shown that equipping primed polyclonal murine CD8+ T cells with micropatches, namely "cellular backpacks (BPs)", can enhance their anti-tumor response against aggressive tumors by providing localized stimulation. These BPs are 6 μm polymeric poly(lactic-co-glycolic acid) (PLGA) microparticle disks functionalized with anti-CD45 antibodies[#Fukuta_Mitragotri_neutrophilBPBrain], which can attach to the cellular membrane of T cells. BPs show strong clinical promise as a companion therapy to extend the persistence of T cell therapies. Therefore, to fully realize their potential, it is essential to understand the diverse ways in which BPs influence T cell behavior, in order to improve the technology and optimize for its clinical application. Previous studies have investigated the biological effects of BPs on immune cells through receptor clustering[#Prakash_2023_BP_NKcells, #Prakash_2024_BP_Bcells] and biophysical interactions[#Ninad_2024_neutrophilBP], as well as their chemical effects for drug and cytokine release[#Kapate_2023_BPMacrophageforTBI, #Kapate_2023_BPMyeloidforMS, #Shields_2020_BPMacrophage]. However, the mechanical impact of BPs on the cytoskeleton and plasma membrane has not been thoroughly explored. Preliminary observations have shown that BPs induce morphological changes and cytoskeletal remodeling in immune cells while still permitting migration, suggesting a unique form of mechanical modulation. The consequences and depth of these effects, however, have remained largely uncharacterized. This thesis specifically has addressed this previously unexplored topic by investigating the impact of BPs on the mechanobiology of T cells. Although interest in T cell mechanosensation has grown in recent years, most studies have focused on the formation of the immunological synapse (IS) and the influence of substrate stiffness, leaving other mechanical aspects of T cell regulation underexamined. BPs provide a unique platform to investigate these questions, as they induce cytoskeletal and morphological changes while maintaining T cell motility and functionality. This work has identified and characterized two well defined aspects of mechanobiology to shed light on how BPs influence T cell mechanobiology, contributing new insights with potential implications for optimizing BP-based immunotherapies. First, the project explored whether BPs could alter the mechanics of physical tumor cell cross-talk with T cells. A 2022 study revealed a new mechanism of immune evasion whereby cancer cells extend tunneling nanotubes (TNTs) structures to pump out the mitochondria of immune cells. This mechanism has been shown to promote premature exhaustion of T cells in the TME, contributing to their lack of efficacy[#saha2022nanotubes]. Exocyst complex proteins of the Sec family and the Rho and Ras GTPase family are known to be involved in actin remodeling during TNT formation and mitochondrial trafficking. The same protein complex is involved in cytoskeletal remodeling due to a mechanical stressor. Hence, this project hypothesized that modifying T cells with cellular BPs would alter this complex from allowing mitochondrial transfer and protect them from mitochondrial theft by cancer cells, thereby prolonging their persistence. We investigated whether BP attachment on T cells would hinder mitochondria transfer by co-culturing them with tumor cells. Although BP attachment to Jurkat T cells was achieved with high efficiency (~80%), it did not impair mitochondrial transfer, suggesting that BP-induced cytoskeletal remodeling may not be sufficient to disrupt TNT formation or that alternative mechanisms of transfer may exist. These results suggest that BPs do not interfere with beneficial intercellular mitochondrial exchange, which could allow T cells to remain metabolically supported in the tumor microenvironment. The second focus investigated whether a biomechanical effect participates in the non-specific activation of T cells caused by BP attachment. Shear stress alone has been shown to activate T cells via the Piezo1 stretch-activated calcium (Ca2+) channel[#sarna2024_ShearStressTcells], and Ca2+ influx is a critical downstream event in T cell receptor (TCR)-mediated signaling. Thus, we hypothesized that the mechanical interaction of the T cell membrane with a BP could trigger Piezo1-mediated Ca2+ entry, thereby promoting an activated T cell phenotype. To test this hypothesis, a pharmacological inhibitor of Piezo1 was applied to T cells, and intracellular Ca2+ levels were monitored. As expected, BP attachment led to elevated intracellular Ca2+ levels, an early marker of activation. However, pharmacological inhibition of Piezo1 did not significantly reduce this Ca2+ influx. Interestingly, longer-term studies revealed that Piezo1 inhibition suppressed the upregulation of the activation marker CD25 and also reduced BP retention on the T cell surface, suggesting that Piezo1 contributes to sustained activation and cytoskeletal stabilization in the context of BP attachment. This indicates that Piezo1 may not initiate the Ca2+ influx but instead plays a role in maintaining T cell responsiveness to sustained mechanical stimuli. In summary, this thesis identified BPs as a unique platform for modulating T cell mechanobiology, revealing both their compatibility with intercellular communication and a potential role for Piezo1 in sustaining mechanical activation of T cells by BPs. These insights lay the groundwork for refining BP-based immunotherapies and uncovering new strategies to support T cell function in solid tumors.